CN111330085A

CN111330085A - Traditional Chinese medicine regulation and control type composite active bone scaffold and preparation method and application thereof

Info

Publication number: CN111330085A
Application number: CN202010124311.4A
Authority: CN
Inventors: 刘昌胜; 陈芳萍; 梁青; 毛丽杰
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2020-02-27
Filing date: 2020-02-27
Publication date: 2020-06-26
Anticipated expiration: 2040-02-27
Also published as: WO2021169759A1; CN111330085B

Abstract

The invention discloses a traditional Chinese medicine regulation type composite active bone scaffold, a preparation method and application thereof, wherein the composite active bone scaffold is formed by compounding an organic component and an inorganic component, and is also loaded with dipsacus asperoides VI, wherein the organic component is selected from PLGA, PLLA, PDLA and POC, and the inorganic component is selected from β -tricalcium phosphate and hydroxyapatite.

Description

Traditional Chinese medicine regulation and control type composite active bone scaffold and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedical materials, relates to a load of active factors and biomolecules and a multi-stage drug release system, and particularly relates to a traditional Chinese medicine regulation type composite active bone scaffold with defect repair and treatment effects.

Background

The health and life quality of human beings are seriously harmed by large-section bone defects (the length of the bone defect is 1.5-2 times larger than the diameter of the bone) caused by trauma, inflammation, infection, tumor, dysplasia and the like, and the repair treatment of the large-section bone defects is a difficult problem which is solved by modern medicine. Along with social development, people suffer frequent accidental injuries due to various problems such as rapid population increase, busy traffic, accelerated life rhythm, increased diseases and natural disasters, sports injury, war and the like, and are accompanied by increasingly more social problems such as large-section bone tissue defect, bone repair and the like. The large bone defect is limited by conditions such as more scar tissues, poor blood circulation, potential infection and the like, is often accompanied with complications such as bone nonunion or short deformity in clinic, has high incidence rate, and brings heavy mental burden and huge economic pressure to individuals, families and society.

Osteoporotic fractures are diseases caused by the disruption of the dynamic balance between osteoblastic bone formation and osteoclastic bone resorption. Among them, the scaffold is one of the key elements for repairing osteoporosis defects, and its composition and structure affect the adhesion and proliferation of cells and the formation of bone tissues.

The dipsacus asperoides is a traditional Chinese medicinal material in Asia, contains important components such as saponin, has the effects of nourishing liver and kidney, continuing fracture and strengthening tendons and bones, and is mainly used for diseases such as limb paralysis, waist soreness and backache, traumatic injury, broken bones and tendons, metrorrhagia and metrostaxis. Although dipsacus asperoides has achieved a certain amount of results in the research on prevention and treatment of osteoporosis, it is still administered mainly by oral administration, and there are few reports on the research on the effect of drugs on bone metabolism at the cellular level. From the perspective of molecular biology and cell biology, it is necessary to intensively study how to enhance the role of drugs or active factors in bone repair.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine regulation and control type composite active bone scaffold which has the advantages of excellent mechanical strength, simple operation, quick forming, controllable pore diameter, degradability, excellent biocompatibility and functions of defect repair and treatment.

The invention provides a composite active bone scaffold, which is formed by compounding an organic component and an inorganic component, and is also loaded with teasel root saponin VI;

wherein the organic component is selected from: polylactic acid-glycolic acid copolymer (PLGA), poly (D-lactic acid) (PDLA), poly (L-lactic acid) (PLLA), poly (citric acid-1, 8-octanediol) ester (POC);

the inorganic component is selected from β -tricalcium phosphate and hydroxyapatite.

In another preferred embodiment, the organic component is preferably PLGA and the inorganic component is preferably β -tricalcium phosphate.

In another preferred embodiment, the PLGA specification is 50:50 lactic acid to glycolic acid; the molecular weight is 3-5 ten thousand.

In another preferred embodiment, the mass ratio of the organic component to the inorganic component is 1:9 to 5:5, preferably 3:7 to 5: 5.

In another preferred embodiment, the mass ratio of the organic component to the inorganic component is 4: 6.

In another preferred embodiment, the mass ratio of the organic component to the inorganic component is 5: 5.

In another preferred embodiment, the optimal ratio of the organic component to the inorganic component is 3:7, in view of its degradability and its mechanical strength.

In another preferred embodiment, the mass ratio of the teasel saponin VI to the inorganic component is 0.1-5%.

In another preferred example, the Chinese medicinal teasel root saponin VI accounts for 0.2%, 0.5%, 1% and 5% of the inorganic component by mass.

In another preferred embodiment, the scaffold is also loaded with BMP-2 and/or 6-O-sulfonated chitosan (26 CSC).

In another preferred embodiment, depending on the size of the scaffold and the amount of BMP-2 loaded in the animal size, 1. mu.g of BMP-2 is instilled per cylindrical scaffold of 10mm diameter and 4mm height at the cellular level, for example.

In another preferred embodiment, the concentration of the PBS solution at 26CSC is 1-10mg/ml, preferably 2-8mg/ml, more preferably 5 mg/ml.

In another preferred embodiment, the pore size of the composite active bone scaffold is 300-500 microns.

In another preferred embodiment, the compressive stress of the composite active bone scaffold at the time of failure is 20-30 MPa.

In another preferred embodiment, the compressive stress at failure of the composite active bone scaffold is 25.41 MPa.

In a second aspect of the present invention, there is provided a method for preparing the composite active bone scaffold of the first aspect, comprising the steps of:

(i) mixing the dipsacoside VI solution and the organic component solution to obtain a mixed solution;

adding inorganic components into the mixed solution to obtain a mixture;

(iii) and 3D printing the mixture or tabletting the mixture to obtain the composite active bone scaffold.

In another preferred example, the concentration of the dipsacoside VI solution is 0.1-0.5 mg/ml.

In another preferred embodiment, the concentration of the solution of the organic component is 1 to 5 g/ml.

In another preferred embodiment, the preparation method comprises the following steps:

(i) dissolving degradable polymer component PLGA with an organic solvent, wherein the proportion of the organic solvent in the polymer is 0.5ml/g, the dissolving temperature is 60 ℃, the water bath heating lasts for 30min, and the magnetic stirrer stirs;

(ii) and (3) fully dissolving the Chinese medicinal teasel root saponin VI in DMSO (1mg/5 mu l), adding the solution in the step (i) and uniformly mixing, adding β -TCP powder into the completely dissolved solution, fully and uniformly stirring to prepare printing slurry with proper viscosity, and printing.

In another preferred example, the 3D printing employs a normal temperature printing nozzle. The printing parameters are as follows: the air pressure is 0.2-0.4MPa, preferably 0.3 MPa; the speed is 2.0-2.5mm/s, preferably 2.0 mm/s; the filling distance is 0.7-0.9 mm. The broken filaments are raised to 0.4-0.8mm, preferably 0.5 mm.

(ii) fully dissolving the Chinese medicinal teasel root saponin VI in DMSO (1mg/5 mu l), adding the solution in the step (i), uniformly mixing, adding β -TCP powder into the completely dissolved solution, fully and uniformly stirring to obtain a dough-like uniform material, twisting into fine strips, cutting into small groups with equal length, and tabletting in a polytetrafluoroethylene mold.

In another preferred embodiment, the solution of the organic component is prepared by dissolving the organic component in 1, 4-dioxane, dichloromethane, chloroform or a mixed solvent thereof.

In another preferred embodiment, the β -TCP is ground with a ball mill and sieved through a 325 mesh sieve with a vibrating sieve machine.

In another preferred embodiment, the β -TCP preparation method comprises the following steps:

respectively preparing Ca (OH) according to the Ca/P ratio of 1.5₂Suspension of (2) and H₃PO₄And (3) solution. At room temperature, 0.6mol/L of Ca (OH)₂The suspension is added dropwise with 0.4mol/L of H₃PO₄And (3) strongly stirring the solution, continuing to react for 5 hours after the dropwise addition is finished, centrifuging and freeze-drying to obtain a precursor, and sintering in a muffle furnace at 900 ℃ for 2 hours to obtain β -TCP.

In another preferred embodiment, the traditional Chinese medicine regulation type composite active bone scaffold with defect repair and treatment functions is placed in an oven with the temperature of 60 ℃ to volatilize the solvent.

In another preferred example, the method also comprises the steps of physical adsorption of 26CSC, dropping BMP-2 after freezing and aseptic drying after saturation of adsorption.

In a third aspect of the invention, the composite active bone scaffold of the first aspect is provided for preparing a repair material or a tissue engineering material for treating bone defects.

In another preferred embodiment, the composite scaffold is prepared for solving the problem of filling and repairing large bone defects caused by trauma, inflammation, infection, tumor, dysplasia and the like and defects of postmenopausal women caused by estrogen deficiency, aging and aggravation of drug side effects.

The composite material of the invention enables the advantages of organic and inorganic components to be complementary and has excellent mechanical property; the Chinese medicinal dipsacus root saponin VI can promote osteogenesis, inhibit osteoclasts and maintain the balance of activities of osteogenesis and osteoclasts; BMP-2 has high osteogenic activity and can participate in the regulation of bone repair process; 26CSC has a function of promoting angiogenesis, thereby promoting osteogenesis. The active factors and biomolecules on the surface of the composite stent are firstly released to play a role in promoting the rapid proliferation of cells, and then the drugs in the stent are slowly released along with the time to provide subsequent force, so that the utilization rate of local drug delivery is improved. The drug-loaded scaffold can obviously promote the proliferation of cells and the expression of osteogenic genes, promote the differentiation behaviors such as osteogenic mineralization and the like, thereby increasing the bone repair capability.

Meanwhile, the preparation method of the organic-inorganic traditional Chinese medicine composite material is simple and rapid, has a regular and controllable pore structure, provides a good microenvironment for the growth of blood vessels for cell growth, and is favorable for adhesion and proliferation of cells.

Within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (e.g., the embodiments) can be combined with each other to constitute a new or preferred technical solution. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 is a digital photograph and SEM picture of a porous composite scaffold.

FIG. 2 is the in vitro NaOH rapid degradation curve (a) and the pH change in Tris-HCl degradation of the porous composite scaffold (b).

Fig. 3 is a result of cytotoxicity evaluation of the composite scaffold.

FIG. 4 is a graph showing the result of ALP activity assay in vitro of Dipsacus asperoides saponin VI.

FIG. 5 is the result of in vitro gene expression analysis of Dipsacus asperoides saponin VI.

Fig. 6 is a graph of compression performance test results.

Detailed Description

The inventor of the application researches extensively and deeply, researches and develops for the first time a traditional Chinese medicine regulation type composite active bone scaffold with defect repair and treatment effects, wherein an organic component of the composite material is preferably poly (lactic-co-glycolic acid) (PLGA), an inorganic component of the composite material is preferably β -tricalcium phosphate (β -TCP), the traditional Chinese medicine regulation type composite active bone scaffold carries traditional Chinese medicine dipsacus asperoides VI, and innovatively compounds traditional Chinese medicine components into the material.

Composite active bone scaffold

The composite active bone scaffold provided by the invention is a traditional Chinese medicine regulation type composite active bone scaffold with defect repair and treatment effects, the organic component of the composite material is preferably poly (lactic-co-glycolic acid) (PLGA), the inorganic component is preferably β -tricalcium phosphate (β -TCP), the composite active bone scaffold also comprises traditional Chinese medicines of dipsacus asperoides VI, BMP-2 and 26CSC (6-O-sulfonated chitosan).

The invention discloses a traditional Chinese medicine regulation type composite active bone scaffold material with defect repair and treatment effects, which is prepared by compounding a degradable polymer PLGA and an inorganic material β -TCP by a solvent method and a traditional Chinese medicine dipsacus asperoides saponin VI, and adopting a 3D printing rapid prototyping technology or a conventional tabletting technology, and simultaneously adsorbing bone morphogenetic protein BMP and an angiogenesis promoting drug 26CSC (6-O-sulfonated chitosan) to be compounded, wherein the traditional Chinese medicine dipsacus asperoides saponin VI can inhibit bone absorption and promote bone formation, has a good anti-osteoporosis effect, plays a role in subsequent bone repair process, has a great clinical application value in promoting bone defect repair caused by osteoporosis and the like, PLGA has good biocompatibility, degradation products are lactic acid and glycolic acid, and are byproducts of human metabolic pathways, and have no toxic or side effect, β -TCP has excellent bone conductivity, is widely used for bone repair materials, but has great brittleness, is difficult to form, and can be combined together by the solvent method to obtain a good performance of the CSC, the composite scaffold 2-bone scaffold has excellent regulation and growth promotion effects of promoting growth of bone morphogenetic scaffold, and growth of a biological extracellular matrix, a biological growth of a biological growth factor, a biological growth of a biological carrier, a biological growth of a biological growth promoting scaffold, a biological growth of a biological growth promoting cell, a biological growth of a biological matrix, a biological growth of a biological growth.

Preparation method

The invention discloses a preparation process of a traditional Chinese medicine regulation type composite active bone scaffold with defect repair and treatment effects.

The preparation process comprises the steps of compounding an organic component and an inorganic component, preparing printing slurry and drying.

β -preparation of TCP powder, respectively preparing Ca (OH) according to Ca/P ratio of 1.5₂Suspension of (2) and H₃PO₄And (3) solution. At room temperature, 0.6mol/L of Ca (OH)₂The suspension is added with 0.4mol/L H according to 1-3 drops/second₃PO₄In the solution, stirring strongly, adjusting the pH value of the solution by using ammonia water in the dropping process, strictly controlling the pH value to be between 7.0 and 7.5, controlling the pH value to be 7.2 after the dropping is finished, continuing to react for 5 hours, centrifuging and freeze-drying to obtain a precursor, sintering for 2 hours in a muffle furnace at 800 ℃ to obtain β -TCP, and sieving with a 325-mesh sieve for later use.

In another preferred example, the molding preparation method of the composite material comprises the following steps:

a) dissolving degradable polymer component PLGA with an organic solvent, wherein the proportion of the organic solvent in the polymer is 0.5ml/g, the dissolving temperature is 60 ℃, the water bath heating lasts for 30min, and the magnetic stirrer stirs;

b) adding β -TCP and radix Dipsaci powder into the completely dissolved solution, stirring to obtain printing slurry with appropriate viscosity, and printing or tabletting.

In another preferred embodiment, the organic solvent is 1, 4-dioxane.

In another preferred embodiment, the inorganic components β -TCP are all milled using a ball mill and sieved through a 325 mesh screen using a vibrating screen machine.

In another preferred example, the teasel saponin VI is dissolved first and then sufficiently dissolved in the above a).

In another preferred example, the 3D printing adopts a normal temperature printing nozzle and a 0.41mm needle head.

In another preferred example, the printing parameters further include model and size design, filling path, extruding pressure, nozzle movement speed, filament outlet distance, filament breakage lifting and the like. The printing model file is in a.stl format; the filling path is linear filling; the extrusion pressure is 0.35 Mpa; the movement speed of the spray head is 3 mm/s; the filament outlet distance is 0.85mm, the layer thickness is 0.2mm, and the broken filaments are raised by 0.5 mm.

In another preferred example, in the step b), the composite scaffold material after being printed and formed is placed in an oven at 60 ℃ to evaporate the solvent.

Use of

The composite scaffold can be used for repairing bone defects and replacing materials and can also be used as a tissue engineering bone.

The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

The invention has the advantages that:

(1) the composite material combines the excellent performances of the organic component and the inorganic component, greatly improves the formability and the mechanical strength of the material, complements the advantages of the organic component and the inorganic component, and obtains the composite bracket material with excellent performance.

(2) The Chinese medicinal dipsacus root saponin VI can promote bone formation, inhibit osteoclast, and maintain the balance of bone formation and osteoclast activity.

(3) The porous material prepared by 3D printing is simple and rapid, has a regular and controllable pore structure, provides a good microenvironment for the growth of blood vessels for cell growth, and is favorable for adhesion and proliferation of cells.

(4) Meanwhile, the forming method is suitable for all similar materials.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless otherwise indicated, percentages and parts are percentages and parts by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1

β preparation of TCP

Respectively preparing Ca (OH) according to the Ca/P ratio of 1.5₂Suspension of (2) and H₃PO₄And (3) solution. At room temperature, 0.6mol/L of Ca (OH)₂The suspension is added with 0.4mol/L H according to 1-3 drops/second₃PO₄In the solution, stirring strongly, adjusting the pH value of the solution by using ammonia water in the dropping process, strictly controlling the pH value to be between 7.0 and 7.5, controlling the pH value to be 7.2 after the dropping is finished, continuing to react for 5 hours, centrifuging and freeze-drying to obtain a precursor, sintering for 2 hours in a muffle furnace at 800 ℃ to obtain β -TCP, and sieving with a 325-mesh sieve for later use.

Example 2

2.1 3D printing, molding and preparing of composite support

a) Dissolving degradable polymer component PLGA with 1, 4-dioxane, wherein the proportion of the 1, 4-dioxane in the polymer is 0.5ml/g, the dissolving temperature is 60 ℃ for heating in water bath for 30min, and stirring with a magnetic stirrer;

b) adding β -TCP powder which is fully and physically mixed uniformly into the completely dissolved solution, fully and uniformly stirring to prepare printing slurry with proper viscosity, and printing, wherein the weight ratio of the organic component to the inorganic component is 3:7-5:5, and the specific printing parameters are shown in table 1.

2.2 preparation of composite bracket carrying Chinese medicine dipsacus root saponin VI by 3D printing forming

a) Dissolving degradable polymer component PLGA with 1, 4-dioxane, wherein the proportion of the 1, 4-dioxane in the polymer is 0.5ml/g, the dissolving temperature is 60 ℃ for heating in water bath for 30min, and stirring with a magnetic stirrer; dissolving radix Dipsaci saponin VI in DMSO (1mg/5 μ l), adding the PLGA solution, and mixing.

b) Adding β -TCP powder which is fully and physically mixed uniformly into the solution obtained in the step a), fully and uniformly stirring to prepare printing slurry with proper viscosity, and printing, wherein the weight ratio of the organic component to the inorganic component is 3:7-5:5, and the specific printing parameters are shown in table 1.

TABLE 1 PLGA/β -3D printing Molding parameters of TCP scaffolds

Example 3

3.1 preforming preparation of composite scaffolds

Dissolving degradable polymer component PLGA with 1, 4-dioxane, wherein the proportion of the 1, 4-dioxane in the polymer is 0.5ml/g, the dissolving temperature is 60 ℃ for heating in water bath for 30min, and stirring with a magnetic stirrer;

adding β -TCP powder which is fully and physically mixed uniformly into the completely dissolved solution, fully and uniformly stirring to obtain a dough-like uniform material, twisting into thin strips, cutting into small groups with equal length, and tabletting in a polytetrafluoroethylene die to obtain the composite material, wherein the ratio of the organic component to the inorganic component is 3:7-5: 5.

3.2 tabletting and forming preparation of composite bracket carrying traditional Chinese medicine dipsacus asperoides VI

Dissolving degradable polymer component PLGA with 1, 4-dioxane, wherein the proportion of the 1, 4-dioxane in the polymer is 0.5ml/g, the dissolving temperature is 60 ℃ for heating in water bath for 30min, and stirring with a magnetic stirrer; (ii) a Dissolving radix Dipsaci saponin VI in DMSO (1mg/5 μ l), adding the PLGA solution, and mixing.

Example 4 adsorption of active factors to composite scaffolds

After the traditional Chinese medicine controlled composite active bone scaffold prepared in examples 2.2 and 3.2 is prepared, cobalt 60 sterilization is performed, 26CSC solution is soaked in sterile condition, physical adsorption is saturated, BMP-2 is instilled, and the bone scaffold is frozen and then is dried in a sterile manner.

The amount of BMP-2 was loaded depending on the size of the scaffold and the size of the animal. Cell level 1. mu.g was instilled per cylindrical scaffold 10mm in diameter and 4mm in height. The PBS solution at 26CSC was 5 mg/ml.

Example 6 composite scaffold morphology and Structure characterization

Taking the blank composite scaffold prepared in example 2.1 as an example, the digital photograph and SEM of the porous composite scaffold are shown as the pore size in fig. 1. The macroscopic aperture and the appearance of the scaffold material are regular, the microscopic surface is rough, and bare inorganic particles exist. The pore size is about 300-500 microns.

Example 7 in vitro degradation Properties of composite scaffolds

Taking the blank composite scaffold prepared in example 3.1 as an example, the change of pH value of the porous composite scaffold degraded in Tris-HCl and the in vitro NaOH rapid degradation curve are shown in FIG. 2. It can be seen that the pH is still maintained around 7.10 at 7 days, while in vitro NaOH rapid degradation curve chart shows that as the polymer proportion is increased, the degradation rate of the composite material is increased, and the final degradation rate is basically consistent with the composite proportion. 3: the 5-day degradation rate of the composite material of 7 reaches 33.22 percent; 4:6, the degradation rate of the composite material reaches 48.06 percent; 5:5, the degradation rate reaches 64.70 percent.

The specific implementation steps of the in vitro Tris-HCL degradation are as follows:

adopting Tris-HCl standard buffer solution as degradation medium, selecting composite bracket as test sample, weighing G1, placing into degradation bottle, soaking bracket according to the ratio of 0.1G/10ml, incubating at 37 deg.C in constant temperature shaking box, changing the solution once every three days, taking out after a certain period of time, and measuring pH value change condition

The in vitro NaOH rapid degradation specific implementation steps are as follows:

in order to obtain the degradability of the composite material in a short time, NaOH with the concentration of 0.1M is adopted, the mass of selected samples is approximately the same, 10ml of NaOH solution is added into each sample, the samples are placed into a constant-temperature shaking box at 37 ℃, the samples are taken out after 1D,2D and 5D respectively, supernatant liquid is poured out, the samples are washed by distilled water, the samples are dried in a vacuum drying box at 40 ℃ overnight, and the samples are weighed, and the degradation rate is calculated according to the G ═ w0-w1)/w0 × 100%.

Example 8 evaluation of cytotoxicity of composite scaffolds

rBMSCs cells were seeded in 24-well plates at a cell concentration of 2 × 10 using CCK-8(Beyotime, shanghai)⁴Cells/well, cultured for 1, 2, 3 days, respectively, the detection step, in which the medium is discarded, 400. mu.l of fresh medium (α -MEM containing 10% FBS) is added to each well, then 40. mu.l of CCK-8 is added to each well (protected from light), the mixture is incubated at 37 ℃ for 2 hours in a shaking flask, the absorbance is measured at 450nm, and the absorbance at 650nm is taken as the background value.

The cytotoxicity of each group was compared with that of the blank control group by taking the synthesized TCP in example 1 and the composite scaffold TCP/PLGA prepared in example 3.1 as examples, and the results are shown in FIG. 3. It can be seen that the synthesized organic-inorganic composite material has excellent cell compatibility.

Example 9 in vitro ALP Activity evaluation

Of the 24 well plates, at 1 × 10⁴The density of cells/well was seeded with rBMSCs cells. BMP-2, 26CSC and Dipsacusasperoides VI were added according to the experimental groups shown in Table 2, wherein the concentrations in the table are the concentrations of factors and drugs in the culture medium. The erythrocyte alkaline phosphatase activity was measured on

days

4, 7 and 14, respectively.

A detection step: the medium was discarded, washed 3 times with PBS, 500. mu.l NP-40 cell lysate was added to each well, and cultured in a shaking flask at a constant temperature of 37 ℃ for 90min to completely lyse the cells. Total protein was determined using the BCA standard curve method. Mu.l of the lysate was taken from each well, placed in a 96-well plate, and 100. mu.l of an alkaline phosphatase working solution (PNPP-Na concentration 1mg/ml) was added thereto and incubated in a shaking flask at a constant temperature of 37 ℃ for 2 hours. The absorbance at 405nm was measured. The final result was 405nm OD/(total protein × incubation time) in units of 405nm OD/mg protein/min.

TABLE 2 ALP Activity analysis Experimental groups

As shown in FIG. 4, the addition of BMP-2 and 26CSC significantly improved the osteogenic mineralization effect compared to the control group, and the addition of Dipsacus asperoides saponin VI at different concentrations was 10 days and 14 days^-5The ALP value of M is highest; particularly, at 14d, the group VI containing the teasel saponin is obviously higher than the group stimulated by only BMP-2 and 26CSC, and the addition of the BMP-2 and the 26CSC can highlight the action effect of the medicament.

Example 10 Gene expression analysis of Dipsacus asperoides Saponin VI

rBMSCs cells were seeded in 24-well plates at a cell concentration of 4 × 10⁴Cells/well, cultured normally for 3 days, and stimulated for 24h with media containing drugs and factors. The experimental groups are shown in table 3.

The detection process includes the steps of firstly, cracking cells, extracting mRNA, preparing a reverse transcription reagent, transcribing the mRNA into cDNA by using PrmeScript RT reagent kit, finally, taking the cDNA obtained through reverse transcription as a template, adding SYBR reagent and upstream and downstream primers, and carrying out amplification detection by an RT-qPCR instrument according to a program, measuring the expression conditions of BMP-2, COL1, OSX, Smad1/4/5, TGF- β 1, VEGF and RANKL genes, wherein the measurement process is divided into 3 stages, the first stage is 95 ℃ and 3min, the second stage is mainly an amplification stage and totally 40 cycles occur, each cycle is 95 ℃ and 10s of temperature reduction to 60 ℃ and 20s of temperature reduction, and the third stage is heated to 95 ℃ again until the program is finished.

As shown in fig. 5, the integrated osteogenesis and osteoclast factor dipsacus saponin VI contributes to the expression of osteogenic genes while inhibiting osteoclasts, thereby promoting the formation of new bone.

By integrating ALP activity detection and PCR expression analysis, the teasel saponin VI (ASP VI), BMP-2 and 26CSC have a synergistic effect, obvious proliferation and mineralization effects, an effect of enhancing ASP VI and an obvious osteogenesis effect.

TABLE 3 ALP Activity analysis Experimental groups

Example 11 mechanical characterization of composite scaffolds

Composite scaffold material prepared as in example 2.1, with compression strength explored, and print scaffold size 10 × 10 × 6mm³The compression property of the stent was measured by a universal material tester and compression was performed at an application rate of 1 mm/min.

In FIG. 6, a is a pair of groups of 10 × 10 × 6mm³The average data of the composite stent in a compression experiment can obtain that the compressive stress of the composite stent in the failure is 25.41Mpa, which is higher than that of a pure β -TCP stent, the average compressive modulus is higher than 21.74Mpa, and the compressive strain is 20.8 percent, which is mainly benefited by the bonding effect of PLGA on inorganic fillers, and the stress-strain curve of the composite stent has no yield platform and is a typical plastic compression curve.

As shown in b-c in FIG. 6, under the same stress condition, the brittleness of the conventional β -TCP stent can be improved by adding PLGA, the pure β -TCP stent becomes powder after being stressed and compressed, while the PLGA/β -TCP composite stent is slightly collapsed in height after being compressed, but the surface pore structure is kept complete, no obvious crack occurs, and the appearance is basically maintained, which shows that the stent can bear larger adjacent tissue stress without being damaged after being implanted in the defect position in the body.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A composite active bone scaffold is characterized in that the composite active bone scaffold is formed by compounding an organic component and an inorganic component, and the scaffold is also loaded with teasel root saponin VI;

wherein the organic component is selected from: PLGA, PDLA, PLLA, POC;

2. The composite active bone scaffold according to claim 1, wherein the mass ratio of the organic component to the inorganic component is 1:9 to 5: 5.

3. The composite active bone scaffold of claim 1, wherein the teasel saponin VI accounts for 0.1-5% of the inorganic component by mass.

4. The composite active bone scaffold according to claim 1, wherein said scaffold is further loaded with BMP-2 and/or 6-O-sulfonated chitosan.

5. The composite active bone scaffold of claim 1, wherein the pore size of the composite active bone scaffold is 300-500 microns.

6. The method for preparing a composite active bone scaffold according to claim 1, comprising the steps of:

adding inorganic components into the mixed solution to obtain a mixture;

7. The method of claim 6, wherein the concentration of the asperosaponin VI solution is 0.1-0.5 mg/ml.

8. The method of claim 6, wherein the solution of the organic component has a concentration of 1 to 5 g/ml.

9. The method according to claim 6, further comprising the steps of physically adsorbing 26CSC, after saturation of the adsorption, instilling BMP-2, freezing, and then drying aseptically.

10. Use of the composite active bone scaffold according to claim 1 for the preparation of a repair material or a tissue engineering material for the treatment of bone defects.